Mitochondrial dysfunction pathologically causes many incurable diseases such as the neurometabolic disease Leigh Syndrome almost certainly resulting in childhood death. It additionally exacerbates most late- onset diseases such as cancer, Alzheimer's, and heart disease. To help fill the unmet medical need for new treatments that prevent the onset of these diseases, the Kaeberlein Group focuses on elucidating novel mechanisms of mitochondrial disease progression and the development of effective pharmacological interventions with specific molecular targets. To do this, we utilize the leading mammalian model of Leigh Syndrome missing the electron transport chain structural protein subunit NDUFS4. These mice exhibit a severe neurodegenerative phenotype and premature death. My group recently discovered the FDA-approved mTOR inhibitor rapamycin can remarkably attenuate disease progression and increase the mean lifespan by ~50% in these mice. Rapamycin also extends lifespan in wild type mice, delays the onset of cancer in cancer-prone mice, improves neurological function in Alzheimer's models, and prevents other hallmarks of aging. We amassed significant evidence that this small molecule remodels the metabolome in brain tissue isolated from NDUFS4- KO mice, including decreased NAD+ levels and a switch from glycolysis to amino acid catabolism. My group recently observed severe deactivation of the mTOR and protein kinase C (PKC) pathways in rapamycin-treated NDUFS4-KO mice by phosphoproteomic analysis. This data revealed an unknown relationship between the mTORC2 and PKC signaling pathways in mitochondrial physiology. I have acquired evidence that inhibition of PKCs extends lifespan in these mice, establishing its role in the pathology of mitochondrial disease. This proposal will characterize this relationship, elucidate its mechanistic implications, and discover new pharmacological interventions to prevent mitochondrial disease progression taking a hypothesis driven approach based on my preliminary data. I will illuminate the role of calcium-dependent signaling in mitochondria-associated ER membranes and probe the importance of individual PKCs in the disease phenotype through chemical and genetic inhibition. Collectively, these complementary basic science studies will provide a better understanding of mitochondrial biology, uncover the role of signaling pathways in mitochondrial disease, and illuminate novel mechanisms of interorganellar communication. The achievement of these aims may even have broad implications in the prevention of diseases of normative aging such as Alzheimer?s, cancer, and heart disease.